Interactions between cytoplasmic myosin and actin are responsible for a variety of cellular motile activities. As in muscle contraction, hydrolysis of ATP by myosin provides the required energy. While the actin-activated ATPase activities of vertabrate smooth muscle and nonmuscle myosins are regulated by phosphorylation, there is disagreement as to the mechanism of this regulation. It has been reported that unphosphorylated gizzard myosin is inactive and that phosphorylation causes a large increase in its actin-activated ATPase activity. However, we have found that unphosphorylated calf thymus cytoplasmic myosin and unphosphorylated calf aorta muscle myosin are active. The main effect of phosphorylation on these myosins is to increase their affinities for actin. At high actin concentrations, the MgATPase activities of thymus and aorta myosins are almost independent of phosphorylation. As it seemed unlikely that actin could stimulate unphosphorylated aorta myosin but not unphosphorylated gizzard myosin, we reexamined the ATPase activity of gizzard myosin. We found that both unphosphorylated and phosphorylated gizzard myosins needed to be filamentous for their MgATPase activities to be activated by actin. Under conditions where the unphosphorylated myosin was filamentious, its MgATPase activities were stimulated 10-fold by actin. Under conditions typically used by other investigators, the unphosphorylated myosin was monomeric, and its MgATPase was not activated by actin. Active unphosphorylated smooth muscle myosin helps explain the ability of smooth muscles to maintain tension in the absence of myosin phosphorylation. We have also used limited proteolysis to identify regions on the heavy chain of thymus myosin which appear to be involved in actin and ATP binding and in the regulation of its interaction with actin.